Hi, if there is no problem of preparation you can increase the contrast by phase-contrast;  is based on converting wave phase differences into amplitude differences and is achieved by under-focusing the objective lens. Another possibility is the use of an energy filter, if you find one suitable machine.

Do you know this paper:

Dganit Danino: Cryo-TEM of soft molecular assemblies; Current Opinion in Colloid & Interface Science 17 (2012) 316–329,

Use this link, if you have free access:

http://ac.els-cdn.com/S1359029412001033/1-s2.0-S1359029412001033-main.pdf?_tid=6a9f8038-5b7e-11e4-9f42-00000aab0f01&acdnat=1414156104_12eaa5d8b93843279773c391bd87f8b3

the contrast of cryo em would be not quite good for small complex.

I am not quite sure that your problem actur

Besides phase plate and energy filter, another way might be lowing the voltage, e.g. 120KeV. Some groups also use some binding agents, such as Fab to improve the contrast with the benefit of orientation determination.

Hi, you may look in the EMDB for a reconstruction of a similar protein: http://www.ebi.ac.uk/pdbe/emdb/. Further, if your sample looks ok in RT neg-staining, you may increase the sample concentration, say 2-10x for cryo. Cryo-negative staining may also help. Sometimes proteins may also be just stuck to the carbon support, with few particles in holes.

Direct Detection Device (DDD) can be one of the solutions for imaging small particles.

Hi Jackie, 360 kDa protein is small protein for cryo-EM. But it is not impossible. I am doing cryo-EM on very small protein, even smaller than your protein. So if you tell me about your condition, may be I can help you. I have few questions about your procedure:

(1) What scope are you using and what is the HT.

(2) What kind of grid are you using (Quantifoil or C-Flat grid)?

(3) Are you using vitrobot for sample freezing? What is your blotting parameter?

(4) You wrote, you are using protein conc. from 20 ug/ml to 20 mg/ml. This is very low concentration to very high concentration. Did you check your sample by negative staining before freezing the cryo sample? What is your dilution for negative staining? How many times higher concentrated sample are you using for cryo in comparison to negative staining?

Hi Somnath, this is a very pleasant surprise - I read your PKS works with great interest, and enjoyed them a lot! Here are the conditions you asked about:

1) Tecnai F20, and HT is 80 kV

2) Quantifoil is used

3)  Yes, a vitrobot is used. Blotting condition was 30-second waiting time, followed by a 1.5-second blot.

4) Yes, I checked my protein using negative stain first. It was 20 ug/mL for negative stain, which produced led to nice distinct particles. For cryo, initially I tried 1 mg/mL for  cryo grids with carbon, but there was no contrast so I increased sequentially all the way to 20 mg/mL.

Thanks for hearing me out, and the encouraging words. It almost seemed like an impossible task, getting contrast for my protein.

360 kDa is plenty big enough to get a low to moderate-ish resolution envelope with a standard 4K CCD - caveats apply. When you say there's no contrast, is this the case if you really ramp down the defocus to say -10 to 15um? Are the particles still invisible then?   I've had similar problems with invisible proteins in the past and I suspect here that you're protein is not going into the ice holes. So it could be sticking to the carbon and hiding there. Or it could be completely repelled off the quantifoil onto the blotting paper. Things to try:

- Do you have any dark, blotchy patches of ice around the holes? Some batches of quantifoils seem to have a hydrophobic contaminant that effects the grid charging and blotting properties.

- Try C-flat grids with a larger spacing. Try continuous blots in the 4 second range.

- Try Graphene Oxide Lacey grids. These are available from Agar and have the benefit  of providing a continuous but very,very thin GO support film above the lacey film. You need a concentration lower than 'normal' cryo, but higher than neg stain and you should be able to get particles in ice that way. These grids need a longer blot than normal (perhaps 12-30 seconds) to thin the ice to a usable level. You can use these grids to see if your particles are on the grid by preparing a cryo grid, screening it in you F20 and then taking the grid out and neg staining it. Can you see particles on the film then? 

Hi Jackie,

It is looking like you have some problems about your concentration. Generally, we are using 10-5 times higher concentration for cryo than negative staining. e.g. If your sample concentration is 0.1 mg/ml, then 10X dilution is okay for negative staining (if your concentration mesurement is not too off ) and negative stain image, you can see clear particle, not very crowded.  For cryo, you have to use 0.1mg/ml, meaning you should not dilute the sample for cryo. 

I have one question, how does your ice look like? If it is too thick, off course you can not see any particle. for thin ice, try this

Q 2/2 grid 1 min glow discharge, 3 ul sample and blotting time 2.5 to 3 sec and defocus range 3.5 um should be fine. 

Hi Richard,

The furthest I've ramped down is -3.5 um, and no more. I've seen dark round objects, but we suspect that they're ice. I've also ordered C-flat grids, so that's the first thing we're gonna try as well. Will try the Graphene Oxide Layer grids if the C-flat grids still don't work.

And hi Somnath,

We used 20 ug/mL for negative, so subsequently we tried 100 ug/mL to 1000 ug/mL, but we still saw nothing as well. My ice seems thin enough, to be honest. We did a 1.5s blot, and the ice isn't thick enough to obscure anything.

Thanks you two for the suggestions! I really appreciate the input. We tried something the other day - the particles first frozen in the cryo grid using vitrobot, and then we let it thaw, followed by negative staining. It was to check whether the particles were getting blotted away in the vitrobot or if it was hiding in the carbon. Turns out the particles are definitely on the grid. We suspect that the particles are virtually invisible in ice, unfortunately. On the brighter side, phase plates are coming in a couple of weeks' time. Will keep this thread update!

Try going progressively further underfocused. If at some point your particles suddenly pop out, then you have thick ice.

It would also be worth preparing a grid with perhaps @0.5% UA final conc included in with your protein to cryo-negatively stain it and up the contrast. If you still can't see it, you don't have it in the ice.

I also talked with a guy in our lab who had a similar problem with a soluble 600kda protein. In his case he found all his protein was sticking to the filter paper and all coming off the grid. It might be worth trying a different filter paper (make your own using the vitrobot paper as a template) such a fine whatman. It might even be worth trying a manual blot in the virtobot using a small piece of filter paper on tweezers (through the side port). Obviously you loose a degree of exact timing, but you can position the paper at 90 degrees to the grid and avoid as much direct contact with the grid : you'd need to blot for 2-3 longer though to get good ice thickness. 

What detector are you using?  Have you properly aligned your microscope (minimized beam tilt, astigmatism)?  What dose are you using? What happens if you take an image with 8x the dose?  Do you see anything then?  Do you see Thon rings in the transform?  Have you tried working at a lower accelerating voltage?  Do you have an appropriately sized objective aperture?  (you should be cutting off signal at your Nyquist frequency, if possible)

Mikes has just reminded me - I meant to mention the Thon rings earlier as part of the thick ice issue. If you can't see at least one clear ring in an image FFT with a normal low dose exposure (~1 sec /

I have no idea how much experience you have with cryo-EM, so feel free to ignore. But I usually start by asking if you can image something easy? Try GroEL which you can purify easily or buy - it is big (800kDa), likes to be in holes and is readily visible at about 1.5um defocus and 15-20e-/Å2. If you get nice pictures of this you can be confident it is something to do with your specimen not you or the microscope!  Having said all that - the other answers ask good questions: how thick is your ice - (try burning a hole in the ice on a carbon part of the grid - the difference in brightness is a quick way to get a feel for this. Are the particles on the carbon/filter paper? Is your concentration right - are particles visible on a neg stain grid?

Hi Jackie, above suggestions sound good. One additional route you could take which worked for one of our proteins is to mix your protein with something else for example TMV. The virus allows you to easily check ice thickness, alignment, data quality etc.... And you can look for your particles in the same view since they will be very different to TMV. 350 is still on the small side so contrast will be an issue, if you have a his tag protein could add a gold label through gold labeled his antibody which will show you where the protein is but can give additional problems in processing. 

Hi Jackie,

What kind of detectors you are using ? Have you been able to fix this issue? Please update the thread if you have solved the issue.

Hi, Jackie. could you please share us how did you solve this problem?

One of the ways to increase the contrast of your specimen is to go for higher defocus (under focus)! But remember that you will lose high resolution signal in your final reconstruction! If you move closer to focus, you will retain high resolution signals (but you will face low contrast issue, sometimes even making particle picking an issue).

Another way is to use Volta Phase plate which increase ms contrast dramatically, and you can move much closer to focus without losing much high resolution information. Best of luck!